Aims: We recently reported that in cultured capillaries, bone-marrow derived mesenchymal stem cells (MSCs) form gap junctional communication (GJC) with endothelial cells (EC), then donate mitochondria to the ECs. Our preliminary data suggest the presence of similar MSC-EC interactions in septic lungs. We will follow up these findings to test the hypotheses that in septic lungs, MSC-EC GJC causes lung retention of intravenously injected MSCs (Specific Aim 1);that the GJC leads to MSC?EC mitochondrial transfer (Specific Aim 2);and that beneficial effects accrue from loss of the receptor (TNFR1) for tumor necrosis factor-a (TNFa) (Specific Aim 2) and lung microvascular barrier enhancement (Specific Aim 3). Procedures: Studies will be developed through two-photon and confocal microscopy of isolated, blood-perfused mouse lungs by means of real-time fluorescent imaging to detect MSC trafficking, microvascular permeability and microvascular TNFR1 expression. GJC will be detected by photolytic uncaging. Mitochondrial transfer will be detected by imaging of fluorescent proteins expressed in MSCs. TNFR1 shedding will be assayed by real-time immunofluorescence. Cytosolic and mitochondrial Ca2+ and cell ROS production will be detected by standard fluorophores, as well by newly available FRET-responsive protein probes. The hypothesis will be tested through expression of mutant proteins, siRNA knockdown and mice lacking specific proteins. Significance: Despite the burgeoning understanding that MSCs are beneficial in acute lung injury (ALI), no studies address fundamental cell signaling mechanisms attributable to MSC interactions with lung microvessels. This project will provide a systematic investigation of these interactions. We will achieve novel understanding of the GJC mechanism underlying MSC?EC mitochondrial transfer as a critical step in MSC protection in ALI. The knowledge gained is likely to be highly relevant to the potential application of MSCs as therapeutic agents in lung disease. PUBLIC HEALTH RELEVANCE: This project aims to determine fundamental mechanisms underlying interactions between intravenously injected, bone-marrow derived mesenchymal stem cells (MSCs) and intact lung microvessels. The findings of this research are likely to impact understanding of mechanisms of acute lung injury and the development of relevant therapy.